Vasily V. Bazhenov

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state (13)C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40-100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state (13)C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The (13)C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of alpha-chitin than beta-chitin.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part I. Isolation and identification of chitin.

Marine invertebrate organisms including sponges (Porifera) not only provide an abundant source of biologically active secondary metabolites but also inspire investigations to develop biomimetic composites, scaffolds and templates for practical use in materials science, biomedicine and tissue engineering. Here, we presented a detailed study of the structural and physico-chemical properties of three-dimensional skeletal scaffolds of the marine sponges Aiolochroia crassa, Aplysina aerophoba, A. cauliformis, A. cavernicola, and A. fulva (Verongida: Demospongiae). We show that these fibrous scaffolds have a multilayered design and are made of chitin. (13)C solid-state NMR spectroscopy, NEXAFS, and IR spectroscopy as well as chitinase digestion and test were applied in order to unequivocally prove the existence of alpha-chitin in all investigated species.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part II: Biomimetic potential and applications

In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts.

Nanostructural organization of naturally occurring composites-part II: silica-chitin-based biocomposites

Investigations of the micro-and nanostructures and chemical composition of the sponge skeletons as examples for natural structural biocomposites are of fundamental scientific relevance. Recently, we show that some demosponges (Verongula gigantea, Aplysina sp.) and glass sponges (Farrea occa, Euplectella aspergillum) possess chitin as a component of their skeletons. The main practical approach we used for chitin isolation was based on alkali treatment of corresponding external layers of spicules sponge material with the aim of obtaining alkali-resistant compounds for detailed analysis. Here, we present a detailed study of the structural and physicochemical properties of spicules of the glass sponge Rossella fibulata. The structural similarity of chitin derived from this sponge to invertebrate alpha chitin has been confirmed by us unambiguously using physicochemical and biochemical methods. This is the first report of a silica-chitin composite biomaterial found in Rossella species. Finally, the present work includes a discussion related to strategies for the practical application of silica-chitin-based composites as biomaterials.

Discovery of 505-million-year old chitin in the basal demosponge Vauxia gracilenta

Sponges are probably the earliest branching animals, and their fossil record dates back to the Precambrian. Identifying their skeletal structure and composition is thus a crucial step in improving our understanding of the early evolution of metazoans. Here, we present the discovery of 505–million-year-old chitin, found in exceptionally well preserved Vauxia gracilenta sponges from the Middle Cambrian Burgess Shale. Our new findings indicate that, given the right fossilization conditions, chitin is stable for much longer than previously suspected. The preservation of chitin in these fossils opens new avenues for research into other ancient fossil groups.

Carboxymethylation of the Fibrillar Collagen With Respect to Formation of Hydroxyapatite

Control over crystal growth by acidic matrix macromolecules is an important process in the formation of many mineralized tissues. Highly acidic macromolecules are postulated intermediates in tissue mineralization, because they sequester many calcium ions and occur in high concentrations at mineralizing foci in distantly related organisms. A prerequisite for biomineralization is the ability of cations like calcium to bind to proteins and to result in concert with appropriate anions like phosphates or carbonates in composite materials with bone-like properties. For this mineralization process the proteins have to be modified with respect to acidification. In this study we modified the protein collagen by carboxymethylation using glucuronic acid. Our experiments showed unambigously, that N(epsilon)-carboxymethyllysine is the major product of the in vitro nonenzymatic glycation reaction between glucuronic acid and collagen. We hypothesized that the function of biomimetically carboxymethylated collagen is to increase the local concentration of corresponding ions so that a critical nucleus of ions can be formed, leading to the formation of the mineral. Thus, the self-organization of HAP nanocrystals on and within collagen fibrils was intensified by carboxymethylation.

Simple Method for Preparation of Nanostructurally Organized Spines of Sand Dollar Scaphechinus mirabilis (Agassiz, 1863)

Unique skeletal formations of marine invertebrates, including representatives of Echinodermata, have the unique potential to serve as templates for bio-inspired materials chemistry, biomimetics, and materials science. The sand dollar Scaphechinus mirabilis (Agassiz, 1983) is widely distributed in the northwest of the Pacific Ocean from southern Japan to the Aleutian Islands. This animal is the main source of naphtochinone-based substances. These compounds have recently drawn medical attention for their use as cardiological and ophthalmological drugs. Unfortunately, after extraction of the naphtochinones, the residual skeletons and spines of the sand dollars were usually discarded. Here, we report the first method for the preparation of nanostructurally organized spines of S. mirabilis, using a simple enzymatic and hydrogen peroxide-based treatment. Application of this method opens the way for development of non-wasteful environmentally clean technology of sand dollars as well-known industrial marine invertebrates.